Non-coding RNA characterization
Roles in X-chromosome inactivation
This paper sought to reveal the mechanisms by
which Xist silences the X chromosome.
what elements are important for exclusion of
transcription machinery and nuclear reorganization?
what are the temporal and spatial characteristics of
Xist-mediated silencing?
Is there a relationship between RNA pol II and Xist RNA accumulation?
Summary of fig 1
- Spatially, transcription machinery excluded from the Xist domain
- Repressive epigenetic modifications are associated with the Xist domain
- Temporally, exclusion is followed by epigenetic chromatin changes
- exlcusion is an early event– differentiation day 1
- epigenetic modification is a later event– day 2
Paradox:
- Prior data shows that X-linked expression falls on day 2-4 of differentiation
(Keohane AM, et al. X-Inactivation and histone H4 acetylation in embryonic stem cells. Dev Biol. 1996 Dec 15;180(2):618-30.)
Resolution:
- Examine lifetime of x-linked RNA transcripts by RNA FISH and RNA pol II IF
Fig 2 summary:
- Exclusion of machinery occurs earlier than repression
- expression persists during differentiation day 1 and 2
- machinery is excluded during the observed expression
- persistence of primary transcripts causes delay in silencing
- Jarid1c escapes complete inactivation because it is distal to Xist locus
- Depletion of Cot-1 RNA signal follows RNA pol II exclusion
- further identifies the temporal relationship between repression and
RNA pol II exclusion
- Genes at the periphery of Xist domain lag in repression
Role of spatial organization in determining txn state
- Xist induced relocalization
- gene position relative to Xist RNA domain
Using 3D reconstruction analysis:
- identify Xist domain via RNA FISH
- identify repressed gene locations via DNA FISH
Fig 3 summary:
-X-linked genes are re-localized toward the interior of the Xist RNA domain
during differentiation
- This re-localization is associated with repression of the genes further into the
Xist domain
Role of Xist A repeats
- ΔA constructs:
- cannot induce silencing
- can coat in cis correctly
- can induce same histone modifications
allows distinction between changes due to Xist
silencing or chromatin modifications
Fig 5 summary:
- A-repeat region not necessary for:
- exclusion of RNA pol II
- repression of Cot-I
- formation of silent nuclear compartment
-delta-A Xist failed to silence X-linked genes
- genes were not re-organized into the silent compartment
Conclusions
- Xist RNA forms a silent nuclear compartment during female ES differentiation
- Txn machinery is rapidly excluded followed by silencing of genes
- Exclusion is proceeded by epigenetic silencing modifications
- Silencing is correlated to position relative to the Xist domain
- Xist domain presents a repressive environment
- Xist induces relocalization of genes to the silent Xist RNA domain
- Function of A-repeat region of Xist
- Not needed for formation of the silent compartment
- Not needed for exclusion of RNA pol II
- Required for gene re-localization
Discussion ?
- Is their measure of RNA pol II exclusion accurate?
- Is fluorescence an accurate enough measure of transcriptional state?
- What about the converse experiment express just the A-repeat region…
- Experiments using truncations of the delta-A construct….
- Does the Xist domain co-localize with the nuclear structure components?
- What is the nature of this repressive RNA “domain”?
- How do Polycomb Group complexes fit with Xist function?
- How does Xist re-organize the X-linked genes into the silent domain?